BRPI0408639B1 - NUCLEIC ACID MOLECULE, VECTOR, YEAST HOST CELL AND METHOD TO PRODUCE HPV31 VIRUS-LIKE PARTICLES - Google Patents

Abstract

nucleic acid molecule, vector, host cell, virus-like particles, method for producing virus-like particles, vaccine, pharmaceutical compositions, and methods for preventing hpv infection and for inducing an immune response in an animal. synthetic DNA molecules encoding the hpv31 l1 protein are provided. specifically, the present invention provides polynucleotides encoding the hpv31 l1 protein, wherein said polynucleotides are free of internal transcription termination signals that are recognized by the yeast. synthetic polynucleotides are also provided that encode hpv31 l1 in which the polynucleotides have been codon optimized for high level expression in a yeast cell. synthetic molecules can be used to produce particles similar to the hpv31 virus (vlps) and to produce vaccines and pharmaceutical compositions comprising hpv31 vlps. the vaccines of the present invention provide effective immunoprophylaxis against papillomavirus infection through antibody neutralizing and cell-mediated immunity.

Description

CROSS REFERENCE WITH RELATED REQUESTS

[001] This application claims the benefit of U.S. Conditional Application No. 60 / 457,172 filed on March 24, 2003, the contents of which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[002] The present invention, in general, concerns, in general, therapy against human papillomavirus (HPV). More specifically, the present invention relates to synthetic polynucleotides encoding HPV31 LI protein and to recombinant vectors and hosts that comprise said polynucleotides. This invention also concerns HPV31 virus-like particles (VLPs) and their use in vaccines and pharmaceutical compositions to prevent and treat HPV.

BACKGROUND OF THE INVENTION

[003] There are more than 80 types of human papillomavirus (HPV), many of which have been associated with a wide variety of biological phenotypes, from benign proliferative warts to malignant carcinomas (for review, see McMurray et al., Int. J. Exp. Pathol. 82 (1): 15 to 33 (2001)). HPV6 and HPV11 are the types most commonly associated with benign warts, non-malignant acuminate condylomata and / or low-grade dysplasia of the genital or respiratory mucosa. HPV16 and HPV18 are the high-risk types most often associated with carcinoma in situ and invasive of the nape, vagina, vulva and canal. More than 90% of cervical carcinomas are associated with HPV16, HPV18 infections or the less prevalent oncogenic types HPV31, -33, -45, -52 and -58 (Schiffman et al., J. Natl. Cancer Inst. 85 ( 12): 958-64 (1993)). The observation that HPV DNA is detected in 90 to 100% of cervical cancers provides strong epidemiological evidence that HPVs cause cervical carcinoma (see Bosch et a., J. Clin. Pathol. 55: 244-265 (2002) ).

[004] Papillomaviruses are small (50 to 60 nm), non-enveloped, icosahedral DNA viruses that encode up to eight early and two late genes. The open reading structures (ORFs) of the viral genomes are designated El to E7 and LI and L2, where "E" denotes precursor and "L" denotes late. LI and L2 encode virus capsid proteins, while E genes are associated with functions such as viral replication and cell transformation.

[005] LI proteins are the main capsid protein and have a molecular weight of 55 to 60 kDa. The L2 protein is a minor capsid protein. Immunological data suggest that most L2 proteins are internal to the Ll protein. Both LI and L2 proteins are highly conserved among different papillomaviruses.

[006] Expression of the Ll protein or a combination of the Ll and L2 proteins in yeast, insect cells, mammalian cells or bacteria leads to the self-assembly of virus-like particles (VLPs) (for review, see Schiller and Roden, in Papillomavirus Reviews: Current Research on Papillomavirus; Lacey, ed. Leeds, UK: Leeds Medical Information, pp 101-12 (1996)). VLPs are morphologically similar to authentic virions and are capable of inducing high titers of neutralizing antibodies when administered to an animal or human. Because VLPs do not contain the potentially oncogenic viral genome, they offer a safe alternative to using live viruses in the development of the HPV vaccine (for review, see Schiller and Hidesheim, J. Clin. Virol. 19: 67 to 74 (2000 )). For this reason, the Ll and L2 genes have been identified as immunological targets for the development of prophylactic and therapeutic vaccines against HPV infection and disease.

[007] The development and commercialization of the vaccine against was impeded by difficulties associated with obtaining high levels of expression of capsid proteins in successfully transformed host organisms, limiting the production of purified protein. Therefore, despite the identification of wild-type nucleotide sequences encoding HPV LI proteins such as HPV31 LI proteins (Goldsborough et al., Virology 171 (1): 306 to 311 (1989), it should be highly desirable to develop a source easily renewable from crude HPV proteins using nucleotide sequences encoding HPV31 LI that are optimized for expression in the target host cell, in addition, it should be useful for producing large quantities of HPV31 LI VLPs having the properties that confer immunity to natural proteins for use in vaccine development.

SUMMARY OF THE INVENTION

[008] The present invention relates to compositions and methods for evoking or enhancing immunity to protein products expressed by the HPV31 Ll genes, which have been associated with cervical cancer. Specifically, the present invention provides polynucleotides encoding the HPV31 Ll protein, wherein said polynucleotides are free of internal transcription termination signals that are recognized by the yeast. Synthetic polynucleotides encoding HPV31 Ll are also provided in which the polynucleotides have been codon-optimized for high level expression in a yeast cell. The present invention further provides HPV31 virus-like particles (VLPs) and discloses the use of VLPs in immunogenic compositions and vaccines for the prevention and / or treatment of HPV cancer associated with HPV.

[009] The present invention relates to synthetic DNA molecules that encode an HPV31 Ll protein. In one aspect of the invention, the nucleotide sequence of the synthetic molecule is altered to eliminate the transcription termination signals that are recognized by the yeast. In another aspect, the codons of the synthetic molecules are designed to use the codons preferred by a yeast cell. The synthetic molecules can be used as a source of HPV31 Ll protein, which can self-assemble into VLPs. Said VLPs can be used in a vaccine based on VLP.

[010] A particular embodiment of the present invention comprises a synthetic nucleic acid molecule encoding the HPV31 Ll protein as shown in SEQ ID NO: 4, said nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[011] As stated above, synthetic polynucleotides encoding the HPV31 Ll gene that are free from the transcription termination signals that are recognized by yeast are provided here. This invention also provides synthetic polynucleotides encoding HPV31 Ll as described, which are further altered to contain the codons that are preferred by yeast cells.

[012] Recombinant vectors and recombinant host cells, both prokaryotic and eukaryotic, are also provided, which contain the nucleic acid molecules throughout this specification.

[013] The present invention relates to a process for the expression of an HPV31 Ll protein in a recombinant host cell, which comprises: (a) introducing a vector that comprises a nucleic acid encoding an HPV31 Ll protein in a host cell of yeast; wherein the nucleic acid molecule is free of internal transcription termination signals that are recognized by the yeast; (b) cultivating the yeast host cell under conditions that allow the expression of said HPV31 Ll protein.

[014] The present invention also relates to a process for expression and an HPV31 Ll protein in a recombinant host cell, which comprises: (a) introducing a vector comprising a nucleic acid encoding an HPV31 Ll protein in a host cell yeast; wherein the nucleic acid molecule is codon-optimized for optimal expression in the yeast host cell; (b) cultivating the yeast host cell under conditions that allow the expression of said HPV31 Ll protein.

[015] In preferred embodiments, the nucleic acid comprises a sequence of nucleotides as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[016] This invention also relates to HPV31 virus-like particles (VLPs), methods of producing HPV31 VLPs and methods of using HPV31 VLPs.

[017] In a preferred embodiment of the invention, HPV31 VLPs are produced in yeast. In another preferred embodiment, yeast is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis and Schizosaccharomyces pombe.

[018] Another aspect of this invention is an HPV31 VLP, which comprises an HPV31 Ll protein produced by an HPV31 Ll gene that is free of transcription termination signals that are recognized by yeast.

[019] Yet another aspect of this invention is an HPV31 VLP, which comprises an HPV31 Ll protein produced by a codon-optimized HPV31 Ll gene. In a preferred embodiment of this aspect of the invention, the HPV31 Ll codon-optimized gene essentially consists of a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[020] This invention also provides a method for producing an immune response in an animal that comprises administering particles similar to the HPV31 virus to the animal. In a preferred embodiment, HPV31 VLPs are produced by a codon-optimized gene. In another preferred embodiment, HPV31 VLPs are produced by a gene that is free of transcription termination sequences that are recognized by yeast.

[021] Yet another aspect of this invention is a method of preventing or treating cervical cancer associated with HPV which comprises administering to a mammal a vaccine comprising HPV31 VLPs. In a preferred embodiment of this aspect of the invention, HPV31 VLPs are produced in yeast.

[022] This invention also relates to a vaccine that comprises particles similar to the HPV31 virus (VLPs).

[023] In an alternative embodiment of this aspect of the invention, the vaccine further comprises VLPs of at least one additional HPV type. In a preferred embodiment, at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV55, HPV56, HPV58, HPV59 and HPV68.

[024] This invention also relates to pharmaceutical compositions that comprise particles similar to the HPV 31 virus. In addition, this invention relates to pharmaceutical compositions that comprise HPV31 VLPs and VLPs of at least one additional HPV type. In a preferred embodiment, at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV55, HPV56, HPV58, HPV59, and HPV68.

[025] As used throughout the specification and the appended claims, the singular forms "one", and "o" include plural references unless the context clearly dictates otherwise.

[026] As used throughout the specification and attached claims, the following definitions and abbreviations apply:

[027] The term "promoter" refers to a recognition site in a strand of DNA to which RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and conduct transcription activity. The complex can be modified by activation sequences called "enhancers" or "upstream activation sequences" or inhibition sequences called "silencers".

[028] The term "vector" refers to some means by which DNA fragments can be introduced into a host organism or host tissue. There are several types of vectors that include plasmids, viruses (including adenovirus), bacteriophages and cosmids.

[029] The designation "wild type 31 Ll sequence" refers to the HPV31 Ll sequence disclosed herein as SEQ ID NO: 1. Although the HPV 31 Ll wild type sequence is described earlier, it is not uncommon to find variations in smaller sequences among the DNAs obtained from clinical isolates.

[030] Therefore, a representative wild-type HPV31 Ll sequence was isolated from previously shown clinical samples containing the HPV 31 DNA (see EXAMPLE 1). The wild type 31 Ll sequence was used as a reference sequence for the comparison of the codon-optimized HPV 31 Ll sequences disclosed in this (see FIGURE 1).

[031] The designation "31 Ll partial reconstruction" refers to a construction, disclosed in this (SEQ ID NO: 2), in which the HPV31 Ll nucleotide sequence has been partially reconstructed to contain yeast's preferred codons for expression great in yeast. Partial 31 Ll reconstruction comprises changes in the middle portion of the wild type HPV 31 Ll nucleotide sequence (nucleotides 697 to 1249). The complete HPV 31 Ll sequence was also reconstructed with preferred yeast codons, which is referred to in this as the "31 Ll total" reconstruction (SEQ ID NO: 3).

[032] The term "effective amount" means sufficient vaccine composition is introduced to produce adequate levels of the polypeptide, so that it results in an immune response. A person skilled in the art will recognize that this level may vary.

[033] A "conservative amino acid substitution" refers to the replacement of one amino acid residue with another, chemically similar amino acid residue. Examples of such conservative substitutions are: replacement of a hydrophobic residue (isoleucine, leucine, valine or methionine) with another; replacement of a polar residue with another polar residue of the same charge (for example, arginine with lysine; glutamic acid with aspartic acid).

[034] The term "mammal" refers to any mammal, including a human being.

[035] "VLP" or "VLPs" means virus-like particle or virus-like particles.

[036] "Synthetic" means that the HPV31 Ll gene has been modified so that it contains a nucleotide sequence that is not the same as the nucleotide sequence present in the naturally occurring wild type HPV31 Ll gene. As stated above, the synthetic molecules that are provided herein comprise a nucleotide sequence that is altered to eliminate the transcription termination signals recognized by the yeast. In this, synthetic molecules are also provided which comprise codons which are preferred for expression by yeast cells. The synthetic molecules provided in this code encode the same amino acid sequences as the HPV31 Ll wild type gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[037] FIGURE 1 is a sequence alignment showing the nucleotides that have been altered in the 31 Ll genes from partial reconstruction (SEQ ID NO: 2) and total reconstruction (SEQ ID NO: 3) (see EXAMPLE 2). The reference sequence is the wild type 31 Ll sequence (SEQ ID NO: 1; see EXAMPLE 1). Nucleotides in the partial and total 31 Ll reconstruction sequences that are identical to the reference sequence are indicated with periods. The altered nucleotides are indicated at their corresponding location. The nucleotide number is contained within parentheses.

[038] FIGURE 2 shows the 31 Ll total reconstruction nucleotide (SEQ ID NO: 3) and amino acid sequences (SEQ ID NO: 4). The nucleotide number is indicated on the left.

[039] FIGURE 3 summarizes the changes between the three constructs in the HPV 31 Ll sequence, which are listed on the left. The fourth column indicates the percentage of nucleotide identity between the indicated construct and the 31 Ll wild type sequence and the fifth column indicates the identity of the amino acid. The last column indicates the number of nucleotides that have been changed as to the yeast's preferred codon sequences and the region where the changes have been made.

[040] FIGURE 4 shows a Northern blot probed specifically for HPV 31 Ll under high stringency (see EXAMPLE 4). Left arrows indicate the position of the truncated and full-length HPV 31 Ll transcripts. Pathways labeled "31 by weight" are from the same yeast RNA preparation containing 31 Ll wild type sequences. The pathway labeled "16" contains HPV16 RNA, which is not recognized by the HPV 31 Ll probe because of the high stringency conditions. The pathway labeled "Neg" is a yeast extract that contains no Ll coding sequence. The pathways labeled "31R" are RNA from two separate isolated colonies that express the 31 Ll partial reconstruction sequence.

[041] FIGURE 5 shows a portion of the data from two capture radioimmunoassay (RIA) experiments in counts per minute (cpm) / mg of total protein (see EXAMPLE 7). The Cpm obtained in the RIA is a relative indicator of HPV 31 Ll VLPs. The RIA data demonstrated an increase in the expression of 31 Ll VLP in yeast extracts and protein from the yeast-preferred codon reconstruction gene sequences.

[042] FIGURE 6 shows a representative sample of the 31 Ll VLPs described in this one, as viewed by electron transmission microscopy (see EXAMPLE 8). The bar represents 100 nm.

DETAILED DESCRIPTION OF THE INVENTION

[043] Most cervical carcinomas are associated with infections of specific oncogenic types of human papillomavirus (HPV). The present invention relates to compositions and methods for evoking or enhancing immunity to protein products expressed by genes of oncogenic HPV types. Specifically, the present invention provides polynucleotides encoding HPV31 Ll and particles similar to the HPV31 virus (VLPs) and discloses the use of said polynucleotides and VLPs in immunogenic compositions and vaccines for the prevention and / or treatment of cancer associated with HPV.

[044] The wild-type HPV31 Ll nucleotide sequence has been reported (Goldsborough et al., Virology 171 (1): 306 to 311 (1989); Genbank Accession # J04353). The present invention provides synthetic DNA molecules that encode the HPV31 Ll protein. The synthetic molecules of the present invention comprise a nucleotide sequence, in which some nucleotides have been altered in order to eliminate the transcription termination signals that are recognized by the yeast. In alternative embodiments, the codons of the synthetic molecules are designed to use the codons preferred by a yeast cell for high-level expression. The synthetic molecules can be used as a source of HPV31 Ll protein, which can self-assemble into VLPs. Said VLPs can be used in a VLP-based vaccine to provide effective immunoprophylaxis against papillomavirus infection through antibody neutralizing and cell-mediated immunity. Such VLP-based vaccines are also useful for the treatment of established HPV infections.

[045] The expression of HPV VLPs in yeast cells offers the advantages of being cost effective and easily adapted for large-scale development in fermenters. However, many HPV Ll proteins, including HPV31 Ll (see EXAMPLE 4), are expressed at low levels in yeast cells. It has been determined in accordance with the present invention that low level expression of HPV31 Ll occurs due to the truncation of RNA transcription that results from the presence of the transcription termination signals that are recognized by the yeast. By altering the HPV31 Ll DNA to eliminate any potential sequences by reassembling the yeast transcription termination sites, it is possible to facilitate full-length RNA transcription resulting in increased HPV31 Ll protein expression.

[046] Consequently, in some embodiments of this invention, changes were made to the HPV31 Ll DNA to eliminate any potential yeast transcription termination sequences. These changes allow expression of the full-length HPV31 transcript, as opposed to a truncated transcript (see EXAMPLE 4), which improves expression yield.

[047] As noted above, the synthetic DNAs of the present invention comprise wild-type HPV31 Ll sequence changes that are made to eliminate the yeast recognized transcription termination sites. A person skilled in the art will recognize that additional DNA molecules can be constructed encoding the HPV31 Ll protein, but not containing yeast transcription termination sites. Techniques for finding yeast transcription termination sequences are well known in the art. Transcription termination and 3 'end formation of yeast mRNAs requires the presence of three signals: (1) an efficient element, such as TATATA or related sequences, that enhances the efficiency of the positioning elements located downstream; (2) the positioning elements, which determine the location of the poly (A) site and (3) the polyadenylation site (usually Py (A) n).

[048] The scientific literature is replete with descriptions of sequences that encode yeast transcription termination signals. See, for example, Guo and Sherman, Trends Biochem. Know. 21: 477 to 481 (1986); Guo and Sherman, Mol. Cell. Biol. 16 (6): 2772 to 2776 (1996); Zaret et al, Cell 28: 563 to 573 (1982); Henikoff et al, Cell 33: 607 to 614 (1983); Thalenfeld et al, J. Biol. Chem. 258 (23): 14065 to 14068 (1983); Zaret et al, J. Mol. Biol. 176: 107 to 135 (1984); Heidmann etal, Mol. Cell Biol 14: 4633 to 4642 (1984); and Russo, Yeast 11: 447 to 453 (1985). Therefore, a person skilled in the art will have no difficulty in determining which sequences to avoid in order to construct a synthetic HPV31 Ll gene that produces a full-length mRNA transcription according to the present invention. In addition, assays and procedures for estimating when a yeast transcription termination sequence is present within the synthetic sequence are well established in the art, so that a person of ordinary skill is able to determine whether a constructed HPV31 Ll sequence comprises the termination strings that need to be eliminated.

[049] As described above, the present invention relates to a nucleic acid molecule encoding HPL type 31 Ll protein, the nucleic acid molecule being free of internal transcription termination signals that are recognized by yeast. In exemplary embodiments of the invention, the synthetic molecules and nucleic acid comprise a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[050] In alternative embodiments of the present invention, HPV31 Ll gene sequences are "optimized" for high level expression in a yeast cell environment. The codon-optimized HPV31 Ll genes covered by the present invention include synthetic molecules encoding HPV31 Ll that are free of internal transcription termination signals that are recognized by yeast, others that comprise at least one codon that is codon-optimized for expression high level in yeast cells.

[051] A "triplet" codon of four possible nucleotide bases can exist in more than 60 variant forms. Because these codons provide the message for only 20 different amino acids (as well as transcription initiation and termination), some amino acids can be encoded by more than one codon, a phenomenon known as codon redundancy. For reasons not fully understood, alternative codons are not uniformly present in the endogenous DNA of different types of cells. In fact, there appears to be a variable hierarchy or "preference" for certain codons in certain types of cells. As an example, the amino acid leucine is specified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA, and TTG. Exhaustive analysis of the codon frequencies of the microorganism genome revealed that the endogenous E. coli DNA most commonly contains the CTG specification codon leucine, while the DNA from yeasts and slime molds most commonly includes a TTA specification codon leucine. In view of this hierarchy, In general, it is believed that the probability of obtaining high levels of expression of a leucine-rich polypeptide by an E. coli host will depend, to some extent, on the frequency of use of the codon. For example, it is likely that a TTA codon-rich gene will be poorly expressed in E. coli, whereby a CTG-rich gene will likely be expressed higher in this host. Similarly, a preferred codon for the expression of a leucine-rich polypeptide in a yeast host cell should be TTA.

[052] The implications of codon preference phenomena in recombinant DNA techniques are manifested and the phenomenon may serve to explain many previous failures to achieve high levels of expression of exogenous genes in successfully transformed host organisms - a less "preferred" codon "may be repeatedly present in the inserted gene and the host cell mechanism may not operate efficiently. This phenomenon suggests that the synthetic genes that have been designated include preferred codons from the designed host cell provide an optimal form of foreign genetic material for the practice of recombinant DNA techniques. Thus, an aspect of this invention is an HPV31 Ll gene that is codon-optimized for expression in a yeast cell. In a preferred embodiment of this invention, it has been observed that the use of alternative codons that encode the same protein sequence can remove limitations on the expression of HPV31 Ll proteins by yeast cells.

[053] According to this invention, the HPV31 Ll gene segments were converted to sequences having identical translated sequences but with the use of alternative codon as described by Sharpand Cowe (Synonymous Codon Usage in Saccharomyces cerevisiae. Yeast 7: 657 to 678 (1991)), which is incorporated into this by reference. In general, a methodology consists of identification codons in the wild-type sequence that are not commonly associated with highly expressed yeast genes and which replace them with optimal codons for high expression in yeast cells. The new gene sequence is then inspected for unwanted sequences by these codon substitutions (for example, "ATTTA" sequences, negligent creation of intron junction recognition sites, unwanted restriction enzyme sites, etc.) , undesirable sequences are eliminated by replacing existing codons with codons that code for the same amino acid. The synthetic gene segments are then tested for improved expression.

[054] The methods described above were used to create synthetic gene segments for HPV31 Ll, resulting in a gene comprising codons optimized for high level expression. While the above procedure provides a summary of our methodology for designing codon-optimized genes for use in HPV vaccines, it is understood by a person skilled in the art that the effectiveness of a similar vaccine or increased gene expression can be achieved by minor variations procedure or minor variations in the sequence.

[055] Accordingly, the present invention relates to a synthetic polynucleotide comprising a nucleotide sequence encoding an HPV31 Ll protein or a biologically active fragment or mutant form of an HPV31 Ll protein, the nucleotide sequence comprising codons optimized for expression in a yeast host. Said mutant forms of the HPV31 Ll protein include, but are not limited to: conservative amino acid substitutions, amino terminal truncations, carboxy terminal truncations, deletions or additions. Any such biologically active and / or mutant fragment will encode a protein or protein fragment that at least substantially mimics the immunological properties of the HPV31 Ll protein as shown in SEQ ID NO: 4. The synthetic polynucleotides of the present invention encode the RNA molecules that express a functional HPV31 Ll protein to be useful in the development of a therapeutic or prophylactic HPV vaccine.

[056] One aspect of this invention is a codon-optimized nucleic acid molecule encoding the HPV31 Ll protein as shown in SEQ ID NO: 4, said nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO: 2 .

[057] Another aspect of this invention is a codon-optimized nucleic acid molecule encoding the HPV31 Ll Protein as shown in SEQ ID NO: 4, said nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO: 3.

[058] The present invention also relates to recombinant vectors and recombinant host cells, both prokaryotic and eukaryotic, which contain the nucleic acid molecules disclosed throughout this specification.

[059] Synthetic HPV31 DNA or fragments of it constructed using the methods described herein can be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcriptional regulatory elements and transferred into prokaryotic or eukaryotic host cells to produce Recombinant HPV31 Ll. Techniques for such manipulations are described in the art (Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1989); Current Protocols in Molecular Biology, Ausubel et al., Green Pub. Associates and Wiley-lnterscience, New York (1988); Yeast Genetics: A Laboratory Course Manual, Rose et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1990), which are incorporated herein by reference in its entirety).

[060] In this way, the present invention also relates to a process for the expression of an HPV31 Ll protein in a recombinant host cell, which comprises: (a) introducing a vector comprising a nucleic acid encoding an HPV31 Ll protein in a yeast host cell; wherein the nucleic acid molecule is codon-optimized for optimal expression in the yeast host cell and; (b) cultivating the yeast host cell under conditions that allow the expression of said HPV31 Ll protein.

[061] The present invention also relates to a process for the expression of an HPV31 Ll protein in a recombinant host cell, which comprises: (a) introducing a vector comprising a nucleic acid encoding an HPV31 Ll protein in a host cell yeast; wherein the nucleic acid molecule is free of internal transcription termination signals that are recognized by the yeast; (b) cultivating the yeast host cell under conditions that allow expression of said HPV31 Ll protein.

[062] This invention also relates to a process for expression and an HPV31 Ll protein in a recombinant host cell, which comprises: (a) introducing a vector comprising a nucleic acid as shown in SEQ ID NO: 2 or SEQID NO : 3 in a yeast host cell and, (b) cultivating a host cell under conditions that allow expression of said HPV31 Ll protein.

[063] The synthetic genes of the present invention can be assembled in an expression cassette comprising the sequences designed to provide sufficient expression of the HPV58 Ll protein in the host cell. The cassette preferably contains the synthetic gene, with related transcriptional and translation control sequences operationally linked to it, such as a promoter and termination sequences. In a preferred embodiment, the promoter is the GALI promoter of S. cerevisiae, although those skilled in the art recognize that any of several other known yeast promoters, such as the GAL10, GAL7, ADH1, TDH3 or PGK promoters or others eukaryotic gene promoters can be used. A preferred transcriptional terminator is the S.Herevisiae ADH1 terminator, although other transcription terminators may also be used. The GAL1 promoter-ADH1 terminator combination is particularly preferred.

[064] Another aspect of this invention is an HPV31 virus-like particle (VLP), methods of producing HPV31 VLPs and methods of using HPV31 VLPs. VLPs can self-assemble when Ll, the major capsid protein of human and animal papillomavirus, is expressed in yeast, exempt cells, mammalian cells or bacteria (for review, see Schiller and Roden in Papillomavirus Reviews: Current Research on Papillomavirus; Lacey, ed. Leeds, UK: Leeds Medical Information, pp 101-12 (1996)). Morphologically indistinct HPV VLPs can also be produced by expressing a combination of the capsid proteins Ll and L2. VLPs are composed of 72 Ll pentamers in an icosahedral structure T = 7 (Baker etal., Biophys. J. 60 (6): 1445-56 (1991)).

[065] VLPs are morphologically similar to authentic virions and are capable of inducing high titers of neutralizing antibodies in administration to an animal. Immunization of rabbits (Breitburd et a!., J. Virol. 69 (6): 3959-63 (1995)) and dogs (Suzich et al., Proc. Natl. Acad. Sci. USA 92 (25): 11553 -57 (1995)) with VLPs has been shown to both induce neutralizing antibodies and to protect against experimental papillomavirus infection. However, because VLPs do not contain the potentially oncogenic viral genome and can self-assemble from a simple gene, they represent a safe alternative to using live viruses in HPV vaccine development (for review, see Schiller and Hidesheim, J. Clin Virol. 19: 67-74 (2000)).

[066] Thus, the present invention relates to virus-like particles comprised of recombinant Ll protein or recombinant Ll + L2 proteins of HPV31.

[067] In a preferred embodiment of the invention, HPV31 VLPs are produced in yeast. In another preferred embodiment, yeast is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, and Schizosaccharomyces pombe.

[068] Another aspect of this invention is an HPV31 VLP, which comprises an HPV31 Ll protein produced by an HPV31 Ll gene that is free of internal transcription termination signals that are recognized by yeast.

[069] Yet another aspect of this invention is an HPV31 VLP comprising an HPV31 Ll protein produced by a codon-optimized HPV31 Ll gene. In a preferred embodiment of this aspect of the invention, the HPV31 Ll codon-optimized gene essentially consists of a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[070] Yet another aspect of this invention is a method of producing HPV31 VLPs, which comprises: (a) transforming yeast with a recombinant DNA molecule encoding the HPV31 Ll protein or HPV31 Ll + L2 proteins; (b) cultivating the transformed yeast under conditions that allow expression of the recombinant DNA molecule to produce the recombinant HPV31 protein and (c) isolating the recombinant HPV31 protein to produce HPV31 VLPs.

[071] In a preferred embodiment of this aspect of the invention, the yeast is transformed with an HPV31 Ll gene that is free of transcription termination signals that are recognized by the yeast. In another preferred embodiment, the yeast is transformed with a codon-optimized HPV31 Ll gene to produce HPV31 VLPs. In a particularly preferred embodiment, the codon-optimized HPV31 Ll gene essentially consists of a nucleotide sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[072] This invention also provides a method for producing an immune response in an animal which comprises administering particles similar to the HPV31 virus to the animal. In a preferred embodiment, HPV31 VLPs are produced by a gene that is free of internal transcription termination sequences that are recognized by yeast. In another preferred embodiment, HPV31 VLPs are produced by a codon-optimized gene.

[073] Yet another aspect of this invention is a method of preventing or treating cervical cancer associated with HPV which comprises administering to a mammal a vaccine comprising HPV31 VLPs. In a preferred embodiment of this aspect of the invention, HPV31 VLPs are produced in yeast.

[074] This invention also relates to a vaccine that comprises particles similar to the HPV31 virus (VLPs).

[075] In an alternative embodiment of this aspect of the invention, the vaccine further comprises VLPs of at least one additional HPV type. In a preferred embodiment, at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV55, HPV56, HPV58, HPV59 and HPV68.

[076] In a preferred embodiment of this aspect of the invention, the vaccine further comprises HPV16 VLPs.

[077] In another preferred embodiment of the invention, the vaccine further comprises HPV16 VLPs and HPV18 VLPs.

[078] In yet another preferred embodiment of the invention, the vaccine further comprises HPV6 VLPs, HPV11 VLPs, HPV16 VLPs and HPV18 VLPs.

[079] This invention also relates to pharmaceutical compositions that comprise particles similar to the HPV 31 virus. In addition, this invention relates to pharmaceutical compositions that comprise HPV31 VLPs and VLPs of at least one additional HPV type. In a preferred embodiment, at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV55, HPV56, HPV58, HPV59 and HPV68.

[080] The vaccine compositions of the present invention can be used alone in appropriate dosages defined by the routine test in order to obtain optimal inhibition of HPV31 infection while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desired.

[081] The amount of virus-like particles to be introduced into a vaccine container will depend on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of about 10 pg to 100 pg and, preferably about 20 pg to 60 pg of VLPs is directly administered into muscle tissue. Subcutaneous injection, intradermal introduction, printing through the skin and other modes of administration, such as intraperitoneal, intravenous or, by inhalation, are also considered. It is also considered that enhancer vaccinations can be provided. Parenteral administration, such as intravenous, intramuscular, subcutaneous or other means of administration with adjuvants such as alum or Merck alum adjuvant, concurrently with or, subsequent to the parenteral introduction of the vaccine of this invention, are also advantageous.

[082] All publications mentioned herein are incorporated by reference for the purpose of describing and disseminating methodologies and materials that can be used in connection with the present invention. Nothing herein should be construed as an admission that the invention is not entitled to meet such disclosure by virtue of the previous invention.

[083] Having described the preferred embodiments of the invention with reference to the accompanying drawings, it should be understood that the invention is not limited to those precise embodiments and that various changes and modifications can be made to it by a person skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[084] The following examples illustrate, but do not limit the invention.

EXAMPLE 1 Determination of a representative HPV 31 Ll sequence

[085] The wild-type HPV 31 Ll sequence has been previously described (Goldsborough et al., Virology 171 (1): 306-311 (1989); Genbank Accession # J04353). It is not uncommon, however, to find minor sequential variations between the DNAs obtained from the chemical isolates. To isolate a representative wild-type HPV31 Ll sequence, DNA was isolated from the three clinical samples previously shown to contain HPV 31 DNA. HPV 31 Ll sequences were amplified in a polymerase chain reaction (PCR) using if Taq DNA polymerase and the following primers: HPV 31 Ll F 5 '- CGT CG A CGT AAA CGT GTA TCA TAT TTT TTT ACA G - 3' (SEQ ID NO: 5) and HPV 31 Ll B 5 '- CAG ACA CAT GTA TTA CAT ACA CAA C - 3 '(SEQ ID NO: 6). The amplified products were electrophoresed in agarose gels and visualized by ethidium bromide pigmentation. The ~ 1500 bp lanes of Ll were excised and the DNA purified using the QIA fast PCR purification kit (Qiagen, Hilden, Germany). The DNA was then ligated to the TA cloning vector, pCR-ll (Invitrogen Corp., Carlsbad, CA), transformed E. coli and placed on LB agar with ampicillin plus IPTG and X-gal for the selection of blue / white colony . The plates were inverted and incubated for 16 hours at 372 ° C. White colonies were cultured in LB medium with ampicillin, shaken at 372 c for 16 hours and minipreps were performed to extract plasmid DNA.

[086] To demonstrate the presence of the Ll gene in the plasmid, restriction endonuclease digestions were conducted and examined by agarose gel electrophoresis and ethidium bromide pigmentation. Sequencing was performed on plasmids containing cloned Ll from each of the three clinical isolates. The translated DNA and amino acid sequences were compared to each other and the HPV 31 Ll Genbank sequences. Sequence analysis of the three clinical isolates revealed that no sequence was identical to the Genbank sequence. The pCR-ll-HPV 31L1 / 81 clone was chosen to be the representative 31L1 sequence and is referred to herein as the "wild type 31 Ll sequence" (SEQ ID NO: 1, see FIGURE 1). The sequence chosen as 31 Ll of the wild type contained a silent substitution in nucleotide 1266 and a change from a C to a G in nucleotide 1295, changing the encoded amino acid from threonine to serine. The 31 Ll total and partial reconstruction genes (SEQ ID NOs: 2 and 3, respectively) also encode a serine at this location (see FIGURE 1). In all cases, the amino acid sequences are identical. The nucleotides were changed in the reconstructed constructs to encode amino acids using yeast-preferred codon sequences and to eliminate potential transcription termination signals (see EXAMPLE 2).

[087] The wild-type 31 Ll sequence was amplified using the LS-101 5'- CTC AGA TCT CAC AAA ACA AAA TGT CTC TGT GGC GGC CTA GC - 3 'primers (SEQ ID NO: 7) and LS -102 5 '- GAC AGA TCT TAC TTT TTA GTT TTT TTA CGT TTT GCT GG - 3' (SEQ ID NO: 8) to add the Bglll extensions. PCR was performed using Vent® DNA polymerase. The PCR product was visualized by ethidium bromide pigmentation on an agarose gel. The ~ 1500 bp range was excised and the DNA was purified using the QIAEX II gel extraction set (Qiagen). The PCR product was then digested with Bg111 at 372 ° C for 2 hours and purified using the QIA fast PCR purification kit. The Bllll digested 31 Ll PCR product was ligated to the Ba7nHI digested pIGLE and E. coli DH5 was transformed. Colonies were evaluated by PCR for HPV31 Ll to insert in the correct orientation. The sequence and orientation were confirmed by sequencing. The selected clone was called dpGAL110-HPV 31L1 # 2.

[088] The maxiprep DNA was then prepared and Saccharomyces cerevisiae was made competent and transformed. The yeast transformation was placed on Leu 'sorbitol top-agar on Leu' sorbitol plates and incubated inverted for 3 to 5 days at 30s C. The colonies were selected and striated for isolation on the Leu sorbitol plates. To induce Ll transcription and protein expression, isolated colonies were subsequently cultured in 5 ml of 5 X Leu 'Ade' sorbitol with 1.6% glucose and 4% galactose in rotating tube cultures at 309 C .

EXAMPLE 2 Yeast codon optimization

[089] Preferred yeast codons have been described (Sharp and Cowe, Yeast 7: 657-678 (1991)). Initially, the intermediate portion of HPV 31 Ll, which represents nucleotides 697-1249, was reconstructed using codons preferred by yeast. The strategy used for reconstruction was to design long-overlapping sense and antisense oligomers that cross the region to be reconstructed, replacing nucleotides with yeast-preferred codon sequences while maintaining the same amino acid sequence. These oligomers were used instead of the DNA standard in the PCR reaction. The additional amplification primers were designed and used to amplify the reconstructed sequences of oligomer patterns with pfu DNA polymerase (Stratagene, La Jolla, CA). The optimal conditions for amplification were specific to the section; however, the most used was a program similar to the following: a denaturation step of 942 C for 1 minute, followed by 15 to 25 cycles of 952 c for denaturing for 30 seconds, 559 C for annealing for 30 seconds, 722 c for extension for 3.5 minutes, followed by 722 c for a final extension for 10 minutes and maintain at 42 C.

[090] PCR products were examined by agarose gel electrophoresis. Strips of the appropriate size were excised and the DNA was gel purified. The amplified fragments were then used as a standard to assemble the intermediate HPV 31 Ll fragment from nucleotide 552 reconstruction. The PCR was then used to amplify nucleotides 1-725 (5 'end) and 1221-1515 (3' end) . A final PCR using the 5 'end, the 3' end and the reconstructed intermediate was performed to generate the partial reconstruction of 31 Ll of total length, referred to in this as the "partial reconstruction of 31 Ll".

[091] The complete 31 Ll sequence has also been reconstructed with the yeast-preferred codons. This construction is referred to in this as the "total reconstruction of 31 Ll". Nine long overlapping oligomers were used to generate yeast 1 to 753 preferred codon nucleotide sequences and four long overlapping oligomers were used to generate yeast 1207 to 1515 preferred codon nucleotide sequences. After amplification and gel purification, these fragments, together with the intermediate reconstructed section described above (nucleotides 697 - 1249), were used together in a PCR reaction to generate the total reconstruction sequence of 31 Ll in total length. This piece was generated with BamHI extensions. The gel-purified, reconstructed 31L1 DNA was digested with BamHI, ligated to BamHI-digested pGAL 110 expression vector and transformed into E. coli DH5 cells. Colonies were evaluated by PCR for HPV 31 Ll insert in the correct orientation. The sequence and orientation were confirmed by sequencing.

[092] Plasmid DNA was prepared. S. cerevisiae cells were made competent and transformed. The yeast was placed on Leu 'sorbitol top-agar on Leu' sorbitol plates and incubated inverted for 3 to 5 days. The colonies were streaked by isolation on Leu 'sorbitol plates. Isolated colonies were subsequently cultured in 5 ml of 5 X Leu-Adebitol with 1.6% glucose and 4% galactose in 309 C rotating tube cultures to induce Ll transcription and protein expression. After 48 to 72 hours, the culture volume equivalent to an OD600 = 10 was pelleted, the supernatant removed and the pellets frozen and stored at -70 ° C.

EXAMPLE 3 Preparation of RNA

[093] The transformed yeast cell pellets, which were induced to express HPV 31 Ll by galactose induction, were thawed on ice and suspended in 1 ml of ice-cold water treated with DEPC. The cells were pelleted by centrifugation and the resulting supernatant was removed. The cell pellet was then resuspended in 400 µl TES (10 mM Tris, pH 7.0.10 mM EDTA and 0.5% SDS). An equal volume of phenol saturated in AE buffer (50 mM NaOAc and 10 mM EDTA) was added. The tube was swirled for 10 seconds and heated to 659 C for 50 minutes with mixing every 10 minutes. The tube was then placed on ice for 5 minutes, followed by centrifugation at 49 C for 5 minutes. The supernatant was collected and transferred to a sterile tube. An additional 400 pl of phenol was added, the tube was whirled, placed on ice for 5 minutes and centrifuged. The supernatant was transferred to a sterile tube and 400 µl of chloroform added, mixed and centrifuged. The supernatant was again collected and transferred to a sterile tube and 40 pl of 3 M Na Acetate, pH 5.2 added, in addition to 1 ml of 100% EtOH. The tube was placed on dry ice for one hour, after which it was centrifuged at high speed to pelletize the RNA. The RNA was washed once with 70% EtOH and air dried. The RNA was then suspended in 100 pl of DEPC-treated water and heated to 652 C for 5 minutes to dissolve. Spectrophotometry was performed to determine the RNA concentration in the sample using the assumption that an A260 reading of 1 = 40 pg / ml RNA when the A260 / 280 is 1.7 - 2.0.

EXAMPLE 4 Northern blot analysis

[094] The initial analysis of yeast expressing wild-type 31 Ll suggested that the HPV 31 Ll protein expression yield was considerably lower than expected. To determine whether the low expression occurred due to a problem in the level of transcription versus the level of translation, Northern blot analysis of HPV 31 Ll transcription was performed. Northern blots were made from gels in which the yeast RNA expressing HPV16 Ll was conducted with yeast RNA expressing HPV31 Ll in the same gel to compare transcription sizes.

[095] A 1.2% agarose formaldehyde gel was molded. Ten micrograms of RNA were combined with denaturation buffer (final concentrations: 6% formaldehyde, 45% formamide and 0.9 x MOPS) and heated to 552 C for 15 minutes. One-tenth volume of gel loading buffer was added and the sample loaded onto gel. Electrophoresis was performed at 65 volts in a 1 x MOPS buffer for ~ 5 hours. The gel was washed for 15 minutes in sterile water followed by two five-minute washes in 10 x SSC. The RNA was transferred to a Hybond-N + nylon membrane (Amersham Biosciences, Piscataway, NJ) by capillary action for 16 hours in 10 x SSC. The RNA was then attached to the nylon membrane by crosslinking using the Amersham crosslinker set to 700 units of energy. After fixation, the nylon membrane was allowed to air dry. The membrane was placed in 30 ml of Zetaprobe buffer at 552 C for 2 hours after which probes labeled by 32 P were added and incubated for 16 hours from 53 to 652 c. The membrane was then washed 3 times in 5 X SSC at room temperature for 20 minutes, followed by 2 times in 0.4 x SSC for 20 minutes at room temperature and once at 609 C for 10 minutes. The probe's DNA was generated by PCR using the specific sense and antisense primers of the HPV 31 Ll sequence. The amplified DNA was labeled by treatment with polynucleotide kinase (PNK) and y-32P ATP at 37 ° C for 1 hour. The stain was rolled up in the "sarem" wrapper and exposed to X-ray film for 16 hours. In the development of the film, probe-hydrated RNA was detected as a black band on the radiograph.

[096] The Northern blot analysis described above revealed that most of the full-length wild type HPV 31 Ll transcripts were considerably less than the total length (see FIGURE 4). However, the 31 Ll partial reconstruction was designed not only to insert the yeast-preferred codons into the middle of the gene, but also to eliminate any potential sequences similar to the yeast transcription termination sites. Northern blot analysis clearly showed that in the reconstruction, the transcription length of the 31 Ll gene was significantly increased to a size corresponding to that of the full length HPV 16 Ll transcript (not shown). In this way, premature transcription termination is likely to be calculated by a significant portion of the low expression yield of the wild-type 31 Ll construction.

EXAMPLE 5 HPV 31 Ll protein expression

[097] Frozen yeast cell pellets from galactose-induced cultures equivalent to OD600 = 10 were thawed on ice and suspended in 300 µl PC buffer (100 mM Na2HPO4 and 0.5 M NaCI, pH 7 , 0) with 2 mM PMSF. 0.5 mm glass beads washed with acid were added, ~ 0.5 g / tube. The tubes were swirled for 15 minutes at 49 C. 7.5 pl of 20% TritonX100 were added and swirled for 5 minutes at 49 C. The tubes were placed on ice for 15 minutes, then centrifuged for 15 minutes at 49 C. The supernatant was transferred to a sterile microcentrifuge tube and stored at -709 C.

EXAMPLE 6 Western blot analysis

[098] Total yeast protein extract from twenty to forty isolated yeast colonies for each HPV 31 Ll construct was analyzed by Western blot to confirm HPV 31 Ll protein expression after galactose induction.

[099] Ten micrograms of the total yeast protein extract were combined with SDS-PAGE loading buffer and heated to 959 ° C for 10 minutes. The proteins were loaded on an 8% SDS-PAGE gel and subjected to electrophoresis in Tris-Glycine buffer. After protein separation, the proteins were transferred to Western gel from nitrocellulose and the stain was blocked in 10% dehydrated non-fat milk in TTBS (saline solution not buffered by Tris with Tween-20) for 16 hours. The stain was washed three times in TTBS. The goat anti-trpE-HPV 16 Ll serum, a polyclonal serum that reacts with HPV 31L1, was applied at a dilution of 1: 1000 in TTBS for 1 hour at room temperature. The spot was washed three times in TTBS and anti-goat-HRP conjugated antibody was applied at a 1: 2500 dilution in TTBS for 1 hour. The spot was washed again three times and ECL ™ detection reagent was applied (Amersham Biosciences, Piscataway, NJ). The autoradiography was then performed. The proteins recognized by the antiserum were visualized by the detection agent as dark bands on the autoradiography.

[0100] In all cases, the HPV 31 Ll protein was detected as a sharp range on the autoradiography corresponding to approximately 55 kD (data not shown). The HPV 16 Ll protein was included as a positive control in the gels.

EXAMPLE 7 Radioimmunoassay (RIA)

[0101] Yeast cells expressing HPV 31 Ll were cultured by a variety of methods, including spin tube cultures, shake flasks and fermenters. The yeast was lysed and the protein extracts manufactured to determine the amount of virus-like HPV31 Ll particles (VLPs) produced per milligram of total protein. To demonstrate HPV 31 Ll VLP expression, a portion of each total yeast protein extract was analyzed by capture radioimmunoassay (RIA).

[0102] The RIA was performed using a monoclonal detection antibody, H31.A6, which is specific for HPV type 31 and specific for conformational VLP. H31.A6 is specific for HPV type 31 Ll as it is observed to bind to intact HPV 31 Ll VLPs and does not recognize denatured HPV 31 VLPs. This mAb can subsequently be detected by an 1125 radio-labeled goat anti-mouse antibody. Therefore, the count values per minute (cpm) correspond to the relative levels of HPV31 Ll VLP expression.

[0103] The polystyrene beads were coated with a polyclonal anti-trpE-HPV31 Ll goat serum diluted 1: 1000 in PBS overnight. The beads were then washed with 5 volumes of sterile distilled water and air dried. The antigen, the total yeast protein extract from isolated yeast colonies, was then loaded into the beads by dilution in PBS with 1% BSA, 0.1% Tween-20 and 0.1% Na Azide and incubated with rotation for one hour. After washing, the beads were distributed one per well in a 20-well polystyrene plate and incubated with H31.A6mAb diluted 1: 50,000 for 17 to 24 hours at room temperature. The beads were washed and goat anti-mouse IgG labeled 1125 was added over an activity range of 23000 to 27000 cpm for 10 pl. After 2 hours, the beads were washed and radioactive counts were recorded in cpm / ml. The base counts of blank reservoirs were subtracted from the total cpm / ml, giving the RIA minus the base value.

[0104] Two experiments were carried out: in experiment 1, the protein extracts of the 31 Ll wild type and the partial reconstruction of 31 Ll were compared and in experiment 2, the protein extracts of the 31 Ll partial reconstruction and the total reconstruction of 31 Ll 31 Ll were compared (see FIGURE 5). The results indicate that the expression of VLP of partial reconstruction of 31 Ll is 6.9 times greater than that of 31 Ll of the wild type. The total reconstruction of 31 Ll has a 1.7 fold increase over the partial reconstruction of 31 Ll. Therefore, 31 Ll expression levels were increased> 7-fold by introducing yeast-preferred codon sequences and eliminating potential transcription termination signals.

EXAMPLE 8 Transmission electron microscopy

[0105] To demonstrate that the HPV 31 Ll protein was in fact self-assembled to form pentameric Ll capsomeres, which in turn self-assembled into virus-like particles, a partially purified 31 Ll total reconstruction protein extract, was subjected to transmission electron microscopy (TEM). The yeast was grown under small scale and pelletized fermentation. The pellets were subjected to purification treatments. The clarified pellet and yeast extracts were analyzed by immunoblot to demonstrate Ll protein expression and retention through the purification procedure. The clarified yeast extracts were then centrifuged on a 45% sucrose pad and the resulting pellet suspended in buffer for TEM analysis (see FIGURE 6). The results indicated that the diameter of the spherical particles in this crude sample varied between 30 and 60 nm with some particles showing a regular arrangement of capsomers.

Claims (8)

1. Nucleic acid molecule, characterized by the fact that it comprises a nucleotide sequence that encodes an HPV31 Ll protein, the nucleic acid molecule comprising a nucleotide sequence defined in SEQ ID NO: 2 or SEQ ID NO: 3. 2. Vector, characterized by the fact that it comprises the nucleic acid molecule defined in claim 1. 3. Yeast host cell, characterized by the fact that it comprises the vector defined in claim 2. 4. Yeast host cell according to claim 3, characterized by the fact that the host cell is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis and Schizosaccharomyces pombe. 5. Yeast host cell according to claim 4, characterized by the fact that the host cell is Saccharomyces cerevisiae. 6. Method for producing HPV31 virus-like particles (VLPs), characterized by the fact that it comprises: (a) transforming yeast with a codon-optimized DNA molecule encoding the HPV31 Ll protein, defined in claim 1; (b) growing the transformed yeast under conditions that allow expression of the codon-optimized DNA molecule to produce a recombinant papillomavirus protein; and (c) isolating the recombinant papillomavirus protein to produce the HPV31 VLPs. 7. Method according to claim 6, characterized by the fact that the yeast is selected from the group consisting of: Saccharomyces Kluyveromyces lactis, and Schizosaccharomyces pombe. 8. Method according to claim 7, characterized by the fact that the yeast is Saccharomyces cerevisiae

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